1. Field of the Invention
A variety of stress-related traits in plants are enhanced by the synergistic effects on abscisic acid (ABA)-inducible gene expression of co-expressed basic leucine zipper (bZIP)-domain transcription factors and B3-domain transcription factors. Additionally, two different B3-domain transcription factors may be used to synergistically regulate ABA-inducible gene expression.
2. Description of Related Art
The growth in the world's population combined with a general increase in global prosperity is creating an increasing demand for food, fiber and sustainable agriculture. It is estimated that the world's population will increase by 80% to 10.8 billion people by 2050, with a concomitant decrease in arable land of 20%. A worthwhile future can only be guaranteed through sustainable agriculture and a protective relationship with nature. For example, rice is the staple food for two-thirds of the world's population and is the primary cereal crop in the world, with worldwide production in 2000 of 600 million tons. 92% of the world's rice is produced in Asia, and 40% of the cultivated area is rain-fed and experiences environmental stress, with losses estimated at 200 million tons/yr. Another example is impacts of drought on maize production in the southwest United States. Based on data from the USDA National Agricultural Statistics Service (http://www.usda.gov/nass/), in 2003 the geographical region spanning southern CA, AZ, NM, TX High Plains and trans-Pecos regions produced 1.2% (127 mil bushels) of the nation's grain corn, valued at >$310 million. It is noteworthy that only 78% of grain corn planted in these regions of the Southwest were subsequently harvested, whereas on average 90% of the planted acreage was harvested throughout the United States. Significantly, for AZ and NM from 1999-2003, the percentages of grain corn harvested ranged from only 35-58%, while the national average over that time period was 90.2%. The basis for these differences between the southwest and the “Corn Belt” harvests, valued at >$48 million in losses last year alone, is due to reductions in yield due to drought stress typically experienced by crops in the southwest. Genetic engineering of maize for increased vegetative drought stress adaptation should result in increased yields and profits for producers. A third example is dryland cotton; estimates of the value-added worth of cotton with increased photosynthetic and water use efficiencies and improved seed qualities exceed $200 million/yr in west Texas, $1 billion/yr in the USA, and $5 billion/yr globally.
Yield enhancement to increase crop production is one of the essential strategies to meet the demand for food by the growing population. In order to supply the world's population in 20 years' time with enough to eat, today's food production will have to be doubled on a third less land and water. For example, due to traditional rice breeding advances, with which germplasm from wild relatives was transferred to cultivated strains, production of rice doubled between 1966 and 1990, but it is estimated that production must increase 60% by 2025 to meet demand. The rate at which growers have been able to further improve crop productivity has declined as improved farming practices have become more fully implemented around the world, as land in developed countries available for conversion to farming has declined, and as concerns about the environmental impact of farming have increased.
While the rate of yield increases from hybrids has slowed in the last two decades, the application of biotechnology and genomics is dramatically increasing innovation in the agricultural and seed industries. Biotechnology as a means of sustainable agriculture is a crucial component to meeting the challenges posed by the interrelated global issues of poverty, hunger, population growth, and environmental degradation in the twenty-first century. Biotechnology enables gene-by-gene analysis and enhancement of crops and is augmenting traditional breeding by enabling faster, targeted development of performance-enhancing traits. These traits currently are designed to create higher-quality animal feed and in the future are expected to include nutritional benefits for humans.
Growers have rapidly adopted the first generation of genetically engineered seed traits, with significant numbers of acres planted. The number of global planted acres of herbicide-tolerant and insect-resistant crops grew from less than 5 million acres in 1995 to approximately 120 million acres in 1999. Despite this rapid growth, the total number of acres covered currently represents only a small fraction of the approximately 3 billion acres of crops cultivated worldwide. Additional growth will come from further adoption of currently available traits and the development of new input and output traits.
Improvement of crop plants for a variety of traits, including disease and pest resistance, adaptation to abiotic stresses, and grain quality improvements such as oil, starch or protein composition, has been achieved by introducing new or modified genes into plant genomes. It has recently been shown that the “Green Revolution” of the 1960s that resulted in large increases in wheat yields was due to adoption of varieties that contain a dominant allele of a gene that controls transcription factor expression by modulating microRNAs, a newly discovered mechanism of gene regulation in mesozoans. Transcription factors control virtually all significant plant traits, including yield, disease resistance, freezing and drought protection, as well as the production of chemicals and proteins used as pharmaceuticals, nutriceuticals and consumer products, by coordinate regulation of multiple target genes whose functions in many cases are not yet known.
The expression of target transgenes and endogenous genes is controlled through a complex set of protein/DNA and protein/protein interactions. Promoters and enhancers can impart patterns of expression that are either constitutive or limited to specific tissues or times during development, or in response to environmental stimuli. There are limitations in the types of expression achievable using existing promoters for transgene expression. One limitation is in the expression level achievable. It is difficult to obtain traits that require relatively high expression of an introduced gene, due to limitations in promoter strength. A second limitation is that the pattern of expression conferred by the particular promoter employed is inflexible in that the same promoter-dependent pattern of expression is conferred from generation to generation. It is desirable to have the ability to regulate trait-conferring transgene expression differently in successive generations. One example would be a trait that has a side effect of being detrimental to seed quality, but which is desired for use in fodder. In this case, it would be desirable to carry the trait-conferring transgene in an inactive state in separate breeding stocks.
Plants are sessile and therefore must perpetually develop in response to their changing environment. Plants have evolved complex, integrated, and overlapping signaling pathways to maintain a plastic growth habit in response to stresses such as drought, salt, cold, as well as hormonal cues such as abscisic acid (ABA). ABA mediates a myriad of physiological processes in growth and development, including cell division, water use efficiency, and gene expression during seed development and in response to environmental stresses such as drought, chilling, salt, pathogen attack, and UV light. Despite the complex multitude of physiological, molecular, genetic, biochemical, and pharmacological data that implicate ABA in stress responses, the adaptive responses to ABA and stresses, and the pathways that trigger them, are largely unknown. Seed maturation and freezing/drought/salt tolerance may have certain protective mechanisms in common, since they share the common phenomenon of dehydration stress.
It would be advantageous for genetic engineering of plants with environmental stress resistance to regulate multiple genes in a particular metabolic or response pathway via a single transgene. Cloning and overexpression of Drought Response Element Binding (DREB)/Cold Binding Factor (CBF) subfamily of the AP2-domain family of transcription factors responsible for cold-inducible gene expression has demonstrated the practical benefits of coordinated activation of uncharacterized gene sets that can confer non-specific protection to transgenic plants by up-regulation or pre-activation of stress-response pathways. The ABA-INSENSITIVE-4 gene (ABI4) is most closely related to the DREB/CBF subfamily of the AP2-domain family. Transgenic overexpression of the transcription factor ALFIN1 enhances expression of the endogenous MsPRP2 gene in alfalfa and improves salinity tolerance of the plants. Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. A multi-component transcription factor/target promoter system that regulates hormone and stress responses could be used to address the limitations of single transgene expression and tap into the natural defense systems of crop plants.
Although hundreds of ABA-regulated genes have been identified to date, many of them homologs from a broad range of species, these are likely to represent a somewhat anecdotal sampling of the full spectrum of ABA-responsive genes. Preliminary genome profiling in Arabidopsis has allowed estimation of the number of plant genes modulated by ABA, with current estimates at about 2000-6000 genes. A number of plant gene products have been identified that may function in desiccation tolerance. The COR genes are cold-, drought-, salt-, and ABA-responsive genes whose protein products are heat stable and hydrophilic; some COR genes have structural similarities to the late embryogenesis-abundant (LEA) proteins. LEA homologues in wheat, maize, barley, carrot, and the resurrection plant Craterostigma plantagineum are induced by ABA and dehydration stress. The exact roles of COR and LEA genes in cold and desiccation tolerance are not yet known, but there is strong evidence that they have an adaptive function in desiccation, freezing, and salt tolerance. Altered expression of ABA signal transduction genes can have beneficial effects on stress adaptation of plants.
The RY-G-box-RY regulatory element is commonly found in seed storage protein gene promoters and is necessary for seed-specific expression of the β-phaseolin and Em promoters. The sequences of the RY-G-box-RY elements that are found in different natural promoters have variations, but can be recognized by the presence of particular nucleotide sequences: CATGCAW (the “RY” feature) and CACGTG (the “G-box”). There is substantial diversity in the cis sequences shown to confer ABA-inducible expression. The smallest promoter units (called ABA-Response Elements; ABREs) that are both necessary and sufficient for ABA induction of gene expression appear to consist of at least two essential cis elements, one of which is usually a G-box and the other a “coupling” element.
A seed-specific regulatory factor, Viviparous-1 (VP1), was first described in 1931 and was cloned by transposon tagging in 1989. The ABA-INSENSITIVE3 (ABI3) gene of Arabidopsis is the genetic equivalent of maize VP1 and was cloned by chromosome walking. VP1/ABI3 is expressed in developing seeds and precedes ABA-inducible storage protein and late-embryogenesis-abundant (LEA) gene expression. Rice and maize protoplasts that transiently overexpress the VP1 cDNA can transactivate ABA-inducible promoters from numerous species. Similar transactivation results have been obtained in homologous transient gene expression systems with the rice VP1 and bean Pv-ALF orthologs. Remarkably, VP1 also has repressor activity towards the germination-specific alpha-amylase genes, but repression is non-cell-autonomous and requires embryo-specific factors other than ABA and VP1.
Structure/function studies with VP1 and PvALF in transient gene expression assays demonstrate that the highly conserved N-terminal acidic domain (A1, VP1 amino acids [aa] 51-163) functions as a transcriptional activator and acts in synergy with ABA. The acidic domain of VP1 is not required for germination-specific alpha-amylase gene repression. The conserved basic B2 region (aa 508-544 of VP1) is required for transactivation of the ABA-inducible Em promoter and for enhancing the in vitro binding of various basic leucine zipper (bZIP) factors to their cognate targets, but not for alpha-amylase gene repression. The B3 domain (aa 632-760) binds specifically to promoter sequences required for transactivation but not to ABA-responsive cis-elements. Furthermore, the B3 domain is not required for synergistic effects of transactivation with ABA or for alpha-amylase gene repression. Pv-ALF facilitates chromatin modification of the ABA-inducible β-Phas promoter, which in turn potentiates ABA-mediated transcription. Carrot and Arabidopsis Pv-ALF orthologs can also direct ABA-inducible seed storage protein expression in leaves when expressed ectopically. The exact molecular mechanisms of ABI3/VP1/Pv-ALF are not known, but the predicted FUS3 and LEAFY COTYLEDON-2 class of regulators that control embryo maturation have a continuous stretch of more than 100 amino acids showing significant sequence similarity to the conserved B3 domains of ABI3/VP1/Pv-ALF. Taken together with genetic results that show FUSCA3 and LEC2 interact with ABI3, these correlations suggest that ABI3, LEC2, and FUS3 may act in partially redundant pathways. The Arabidopsis genome encodes 43 members of the B3-domain family, 19 of them within the ABI3/VP1-related subfamily, and their functions are largely unknown.
There are 81 predicted bZIP factor genes in Arabidopsis, but only one bZIP subfamily has been genetically or functionally linked to ABA response: that composed of ABI5 and its homologs, including the ABRE Binding Factors (ABFs and AREBs), Enhanced Em Level (EEL/AtbZIP12), and AtbZIP13-15, 27, and 67, which include the AtDPBFs (Arabidopsis thaliana Dc3 Promoter Binding Factors). Homologs of these genes have been characterized in sunflower and rice, where they are also correlated with ABA-, seed- or stress-induced gene expression. However, studies of bZIPs from other species have shown that in vitro binding of ABREs need not reflect action in ABA signaling in vivo. A rice homolog of ABI5, TRAB1, was identified by a yeast two-hybrid screen using the basic domains of OsVP1 as “bait” and shown to interact with ABREs in vitro and activate ABA-inducible transcription in rice protoplasts. AREBs and ABFs both share with AB15 three conserved charged domains (C1-C3) in their amino-halves as well as the bZIP domain and another conserved (C4) domain at the C-terminus. In vitro studies with the DPBFs and other AB15-family members have demonstrated that this subfamily binds to G-box promoter elements (ABREs) required for ABA regulation. However, the ABI5/DPBF/ABF/AREB subfamily has a broader consensus sequence for its binding site than the other bZIP proteins in that its members tolerate variability in the ACGT core element essential to the ABRE G-box. ABI5 and its homolog DPBF4/EEL were shown to compete for the same binding sites in the AtEm1 promoter and a model was proposed, based on single and double mutant phenotypes of altered gene expression, that EEL directly antagonized ABI5 transactivation. Analyses of transcript accumulation in abi5 mutants suggest that, similar to ABI3, ABI5 has both activator and repressor functions, but that ABI5 and ABI3 may have either synergistic or antagonistic effects on gene expression, depending on the gene. ABI5 protein accumulation is further enhanced by ABA-induced phosphorylation and resulting stabilization of the protein, at least during the early phases of germination.
As indicated above, it has been previously shown that maize VP1 is functionally redundant with ABI3 and that other orthologues of VP1/ABI3 could substitute for VP1 in a multi-component heterologous transactivation system. Consistent with this, expression of an Arabidopsis GIBBERELLIN-INSENSITIVE (GAI) orthologue (the same gene responsible for the “Green Revolution” in wheat; see above) in transgenic rice resulted in desirable dwarfing traits, suggesting that heterologous regulatory genes can be used to affect traits in a wide range of crop species. Transgenic rice plants that express the maize phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) exhibit a higher photosynthetic capacity (up to 35%) than untransformed plants, mainly associated with an enhanced stomatal conductance and a higher internal CO2 concentration. An additional benefit of using heterologous genes is that they may minimize artifacts such as co-suppression and posttranscriptional transgene silencing.
Coordinated regulation of multiple endogenous genes is important for stress adaptation. New methods which genetically engineer value-added vegetative traits for stress adaptation and seed qualities by directed expression of ABA-related transcription factors would be beneficial to supply the world with the increased amounts of food needed by future generations. By overcoming the limitations of targeted gene expression and by transactivation of endogenous plant stress adaptation pathways, the volume and quality of plant products, especially from environments under stress, will be improved.